Ken-Ichiro Yamada

Development and Evaluation of Fluorogenic TaqMan Reverse Transcriptase PCR Assays for Detection of Dengue Virus Types 1 to 4

ABSTRACT The fluorogenic TaqMan reverse transcriptase PCR (RT-PCR) assay was developed for detecting each of the dengue virus (DV) types 1 to 4. DV genome was detected in all the 35 serum samples from confirmed dengue cases by the TaqMan RT-PCR, although it was not detected in 13 and 21% by conventional type-specific and cross-reactive RT-PCR, respectively.

Virus isolation as one of the diagnostic methods for dengue virus infection

Virus isolation is the most reliable evidence of infection. In the present study, we isolated virus from serum samples collected from confirmed dengue cases. When data were analyzed based on disease days, dengue viruses were isolated from 28 of 32 serum samples collected on disease day 5 or earlier. When analyzed based on fever days, dengue viruses were isolated from all the serum samples collected on fever day -3 or earlier, and from 10 of 13 samples collected on fever days -2 and -1. Viruses were isolated from one each of the serum samples collected on fever days 0, 1, 2 and 3, respectively. Virus was not, however, isolated from those collected on fever day 4 or later. The results of virus isolation and reverse transcriptase-polymerase chain reaction were consistent in 78 of 82 serum samples. These results suggest that virus isolation is a useful and sensitive technique for confirmation of dengue virus infection, especially when serum samples are collected before fever subsides.

Partial protective effect of inactivated Japanese encephalitis vaccine on lethal West Nile virus infection in mice.

The effect of mouse brain-derived, inactivated Japanese encephalitis (JE) vaccine on West Nile virus (WNV) infection was examined using a murine model. Mice were immunized with JE vaccine twice and challenged with lethal doses of WNV. When mice were intracranially challenged with WNV, none of the immunized mice were protected. When mice were intraperitoneally challenged, the immunized mice were protected at higher immunization levels. Cross-reactive neutralizing antibodies to WNV were induced by immunization with JE vaccine; however, the levels were much lower than those to JEV. These results indicate that the currently available mouse brain-derived inactivated JE vaccine can induce partial protective immunity to WNV in mice.

Nucleoprotein gene analysis of fixed and street rabies virus variants using RT-PCR

SummaryA simple and rapid single-step reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the nucleoprotein (N) gene of 11 rabies viruses. A conserved set of RT-PCR primers was designed to amplify the most variable region in the N gene. N gene regions were amplified from 6 fixed laboratory viruses, 4 street viruses from dogs in Thailand, and a horse in Zambia. Sequences of the amplified products, together with the database of 91 additional sequences, were analyzed by using PILEUP program of the GCG package. The rabies viruses grouped into at least 9 distinct clusters by <90% nucleotide similarity of the N gene region: I (4 isolates, USA), II (2 isolates, South America), III (3 isolates, Africa), IV (52 strains, Europe, Middle East, Africa and South America), V (16 isolates, North America and Arctic), VI (17 isolates, Africa), VII (1 isolate, Africa), VIII (6 isolates, Thailand and Malaysia) and IX (1 isolate, Sri Lanka). A unique group of rabies viruses from Thailand and clusters of isolates corresponding to their geographic origin also were determined. The simple and rapid single-step RT-PCR proved to be useful for identifying rabies viruses, and for grouping the viruses into clades by sequence analysis.

Serotype-Cross-Reactive Immunoglobulin M Responses in Dengue Virus Infections Determined by Enzyme-Linked Immunosorbent Assay

ABSTRACT We developed immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assays (ELISAs) with four monovalent dengue virus antigens. We attempted to determine whether IgM responses in dengue virus infections are serotype specific or serotype cross-reactive. Serum samples from 14 confirmed dengue cases were examined. In these 14 cases, which consisted of 12 Japanese and 2 non-Japanese patients, infecting dengue virus serotypes were defined by reverse transcription-PCR. Thirteen of the 14 cases were IgM positive in ELISA. IgM responses were serotype cross-reactive in these 13 cases but were highest against infecting dengue virus serotype in 9 of the 13 cases. These results indicate that IgM responses are generally dengue serotype cross-reactive but that IgM levels are highest against the infecting serotype in most dengue cases.

Antibody Responses Determined for Japanese Dengue Fever Patients by Neutralization and Hemagglutination Inhibition Assays Demonstrate Cross-Reactivity between Dengue and Japanese Encephalitis Viruses

ABSTRACT Titers of antibodies to infecting dengue virus serotypes determined by serum neutralization assay were higher than those of antibody to Japanese encephalitis (JE) virus in Japanese dengue patients after disease day 8. Titers of antibody to dengue virus antigens determined by hemagglutination inhibition (HI) assay were higher in only 1 of 23 serum specimens after disease day 11. Thus, the neutralization test is more reliable than the HI test for serological diagnosis of dengue in countries where JE vaccination is widely used or JE is endemic.

Antibody responses induced by immunization with a Japanese rabies vaccine determined by neutralization test and enzyme-linked immunosorbent assay

The immunogenicity of a Japanese purified chick embryo cell culture rabies vaccine (PCECV) was examined. Serum samples were obtained from 86 subjects after pre-exposure or post-exposure prophylaxis. Rabies antibody titres were determined by neutralization test and enzyme-linked immunosorbent assay (ELISA). Titres higher than 0.5 international units (IU)/ml were demonstrated by neutralization test in all the 19 subjects after three-time pre-exposure immunization on days 0, 30 and 180. Titres higher than 0.5IU/ml were also demonstrated by neutralization test in all the 23 subjects after four-time post-exposure immunization on days 0, 3, 7 and 14. There was a correlation between neutralization and ELISA antibody titres (r=0.697); however, neutralization titers were higher than ELISA titres for most of the samples. The results suggest that current Japanese rabies vaccine induces recommended levels of neutralizing antibodies after pre- and post-exposure prophylaxes.